High occurrence of Blastocystis sp. subtype 3 in individuals referred to medical laboratories in Kermanshah, Iran

Aim: The current study investigated the prevalence and genotypes of Blastocystis sp. in individuals who referred to medical laboratories in Kermanshah, Iran. Background: Blastocystis sp. is a common intestinal protozoan found in humans and a wide range of animals, and it is involved in the development of gastrointestinal disorders. Methods: A total of 950 stool samples were examined using the standard formalin-ether concentration technique. All specimens were cultured in Robinson xenic medium. Subsequently, DNA extraction and PCR amplification of subtype specific sequence-tagged site (STS) were conducted. Results Microscopic examination showed that 86 out of 950 samples (9.05%) were infected with Blastocystis sp. Subsequently, 33 of 86 positive samples were cultured and molecularly confirmed by conventional PCR, indicating six subtypes (ST1-ST6). Of note, ST3 (45.0%) was the predominant subtype, followed by ST1 (15.15%) and ST5 (12%). Conclusion: Based on the current findings, ST3 was the most frequent subtype among all positive samples. Having a better understanding of Blastocystis sp. subtype distribution and risk factors would lead to improved preventive measures.

Introduction 1 Blastocystis sp. is a commonly found protozoan parasite of the intestinal lumen of insects and a wide range of vertebrates, including humans (1)(2)(3). Blastocystis sp. is transmitted through the fecal-oral route (infected food or water by cyst) (4,5). Close contact with animals also represents a major predisposing factor for Blastocystis sp. transmission (6,7). Despite its global prevalence, Blastocystis sp. infection has been frequently reported from developing nations. Several risk factors have been recognized in these areas, including inadequate access to safe to accurately determine the parasite and its subtypes; hence, their use is limited. Culture medium, on the other hand, is more sensitive (18)(19)(20). Molecular methods have been extensively developed for the classification of Blastocystis sp. subtypes. Based on a recent molecular study on the small subunit ribosomal RNA (SSU-rRNA) or18S rRNA gene, Blastocystis sp. has been sorted into at least 26 subtypes (ST), and ST1-ST4 have been highly reported in humans (21,22). Reportedly, specific STs may contribute to degrees of pathogenicity, although with conflicting results which demand further exploration. (9).
According to epidemiological studies, ST1-ST8 along with ST12 are of zoonotic concern, ST9 is exclusively anthroponotic, and ST10, ST11, and ST13-ST26 have been only identified in animals (22)(23)(24). Studies aimed at Blastocystis sp. subtyping would inevitably expand our knowledge on geographical distribution, host specificity, transmission routes, and pathogenicity of this commonplace parasite (25)(26)(27). In Kermanshah province, located in the west of Iran, no studies on genetic diversity of Blastocystis sp. have been conducted in human population. The current study aimed to assess the prevalence and genetic diversity of Blastocystis sp. isolates from humans in Kermanshah using the polymerase chain reaction (PCR) method and employing seven pairs of STS primers along with two pairs of ST-specific primers.

Study area
Kermanshah city (34°18′N, 47°3′E), the capital of Kermanshah province, is the largest city in the west of Iran with one million inhabitants. The city is located 1420 meters above sea level and possesses a mountain climate with an annual average rainfall of 500 millimeters (28).

Sample collection and parasitological examination
The present cross-sectional study was conducted on a total number of 950 individuals who referred to the medical laboratories of Kermanshah University of Medical Sciences. The stool specimens were collected in a plastic container without fixative and sent to the parasitology laboratory for microscopic examination.

Microscopic method
All stool samples were concentrated using the formalin-ether technique and examined using Lugol's iodine staining. Next, a cover slip was placed on the surface of each prepared sample, and the samples were examined by light microscopy at a magnification of
When the typical vacuolar or granular forms of Blastocystis sp. organisms were observed, they were sub-cultured in a new medium. The cultivated samples were examined every 48-72 h by iodine staining under a light microscope. Negative samples were checked for 5 consecutive days to confirm the negative result.

Extraction of Genomic DNA
DNA extraction was performed on the pure Blastocystis sp. cultures, using QIAamp DNA Stool mini kit (cat. no. 51504) according to the manufacturer's instructions (30).

Subtyping
To determine the Blastocystis sp. subtype, we used seven pairs of STS (subtype specific sequence-tagged site) and two pairs of ST-specific primers (Table 1) (31,32). The PCR reaction included 7.5 μL of 2X Taq DNA polymerase Mix Red-Mgcl2 (Ampliquon©, Denmark), 3 μL of extracted DNA, 1 μL of each primer (10 p mol), and 2.5 μL of sterile water. The PCR reaction was performed in a final volume of 15 μL.
PCR conditions for the nine primer pairs included an initial denaturation step at 94 ℃ for three min, followed by 35 cycles of including denaturation at 94 ℃ for 30 sec, annealing at 56 ℃ for 30 sec, extension at 72 ℃ for 30 sec, and a final extension step at 72 ℃ for five min. Subsequently, PCR products were loaded into wells of a plate with 1.5% agarose gel containing CinnaGen DNA safe stain and visualized using a UV transilluminator. The size of the amplicon was determined using a 100 bp DNA marker and represented the parasite subtype (Table 1).

Discussion
The parasites of the genus Blastocystis sp. demonstrate a high rate of genetic diversity and varied host specificity worldwide (9). Epidemiological studies have reported different prevalence rates in examined human populations, which could be attributed to demographic settings such as cultural habitats, sanitation levels, the socioeconomic status of the affected people, rural living areas, exposure to animal reservoirs, and diagnostic method (8,9). Before 2013, molecular analysis of SSU rRNA locus using STS primers revealed 17 subtypes, while ST18-ST26 were proposed after 2013. Later in 2020, Stensvold et al. rejected STs 18-20 and ST22 according to the guidelines of Blastocystis sp. subtyping and recommended keeping ST21 and STs 23-26 until further examination (9,24).
In the present study, Blastocystis sp. was detected in 86 (9.05%) of the 950 stool specimens examined by routine diagnostic method (formalin-ether technique). To prevent misdiagnosis due to microscopy, eliminate stool inhibitors, and provide pure parasites, the culture method was preferred (33,34). In addition, it's possible that some Blastocystis sp. subtypes do not grow or grow slowly in axenic culture (18). According to Robert et al., the PCR method is a highly sensitive method (94%) for detecting Blastocystis sp., compared with microscopy (48%) (18).
In the present study, 33 microscopy-positive samples were grown on Robinson culture medium, all of which were subtyped using the PCR method. Previously, several studies employed STS primers, ITS sequencing, PCR-RFLP, and Real-time PCR to identify Blastocystis sp. subtypes in Iran (6,35,36).
Although ST3 demonstrates strict host-specificity to humans, it has rarely been isolated from primates, pigs, dogs, cattle, or rodents (21,49,50). Reportedly, symptomatic and asymptomatic people are prone to this subtype, while other studies have denied an association between Blastocystis sp. and gastrointestinal symptoms (26,37,(51)(52)(53). In this sense, El Safadi et al. (2014) showed ST3 to be a seemingly frequent subtype in the symptomatic group (54). ST1 has been said to have low host specificity and has been isolated from humans as well s a range of animals including monkeys, chickens, cattle, pigs,  dogs, and non-human primates (55,56). In contrast to the current findings, ST1 was markedly prevalent in other parts of Iran (Qazvin and Hamadan), Libya (Africa), Thailand (Asia), Brazil and Colombia (Latin America) (9,55,(57)(58)(59)(60). These observations highlight large-scale inter-human transmission (9,61). Zoonotic transmission of ST1 in the Kermanshah population is a possibility, because some families live in the suburbs near farm animals. Nevertheless, no investigation has been done on animal hosts in this area. Salehi et al. (2020) conducted a study on the prevalence and distribution of Blastocystis sp. subtypes in non-human hosts (poultry, sheep, and cattle) in the southwest region of Iran and reported a prevalence rate of 29.1% in animal hosts; ST1 was also confirmed in two cattle samples. Another study in Khorramabad, in western Iran, ST1 was not found in cattle. In a recently published paper, Rahimi et al. detected Blastocystis sp. in cattle, sheep, and chicken samples and confirmed ST1 in cattle (6,7,27).  emphasized that animal infection with a particular subtype may not reasonably justify its presence in humans (9).
Regarding the isolation of ST5, in four single and four mixed specimens, zoonotic transmission is a possibility, because cattle and pig are dominant reservoir animals for this subtype. Cattle husbandry around Kermanshah may be considered a reason for the presence of a zoonotic cycle. Previously, this subtype was reported from human and livestock in the south, west, and southwest of Iran (7,27,36,(62)(63)(64).
Blastocystis sp. ST2 is a major subtype globally, and has been found to be the most frequent subtype in some studies in Iran (Tehran and Shiraz) and other parts of the world, such as Turkey, Spain, and Bolivia (62, 65-69, 59, 70). In contrast, the current study detected ST2 only in a sample with mixed infections.
Although rodents are reservoir hosts for ST4 and are found all over the world, this subtype is most common in Europe. This may be due to the lack of information in many Asian countries and Africa. In an Iranian study by Mohammadpour et al. (2020), ST4 was isolated from cats and rats in Fars province, in southern Iran (75).
Birds are commonly infected with ST6, which has also been documented in livestock and humans (23). The current results showed the presence of ST6 in Kermanshah people, as observed in three mixed samples. Previously, this subtype was reported in humans, poultry, and cattle in the southwest, west, and south of Iran (24,49,55,64). Of note, ST7, ST8, and ST9 were not identified in human population of Kermanshah.
Overall, several Blastocystis sp. subtypes can be found in human fecal samples simultaneously, and such mixed infections may result from various sources of infection (40). According to different studies, the prevalence of mixed infections ranges 1.1-14.6%, whereas herein, a higher prevalence was found (27%) (57). Moreover, the current study characterized ST1/ST3 and ST5 in mixed infections from Kermanshah, while other Iranian studies have reported ST1 and ST3 in them (57,64,76). A major finding of the current study was that some rare subtypes were distinguished in mixed infections, comparable to other studies across the country (26,36,57). In total, the evaluation of mixed infections and relevant subtypes is a cumbersome procedure and requires pure parasite cultures and molecular identification using subtyping methods (40).
To the best of the authors' knowledge, this is the first molecular study to investigate the distribution of human Blastocystis sp. subtypes in Kermanshah, Iran. Evidence of the molecular epidemiology of Blastocystis sp. in all parts of Iran is insufficient. In this study, ST3 was the most common subtype, followed by ST1, ST5, ST6, and ST4. Further studies are needed to identify Blastocystis sp. subtypes in different hosts. Recognition of Blastocystis sp. subtype distribution may help our understanding of the associated risk factors and transmission routes. Overall, the findings represented here indicate the possibility of zoonotic transmission of Blastocystis sp. in Kermanshah city.

Acknowledgment
This study was part of Bahman Maleki's PhD thesis, which was financially supported by Tarbiat Modares University's Vice-Chancellor for Research. The authors thank the Vice Chancellor for Research of Tarbiat Modares University for their support of this study.